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• W2943383566 abstract "Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4−/−). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4−/− mice. We noted similar results in mice infected with adeno-associated virus–expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA–mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4–ceramide–PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders. Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4−/−). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4−/− mice. We noted similar results in mice infected with adeno-associated virus–expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA–mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4–ceramide–PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders. Chronic exposure to glucocorticoids has long been associated with the development of lipid disorders including dyslipidemia and hepatic steatosis (1Woods C.P. Hazlehurst J.M. Tomlinson J.W. Glucocorticoids and non-alcoholic fatty liver disease.J. Steroid Biochem. Mol. Biol. 2015; 154 (26241028): 94-10310.1016/j.jsbmb.2015.07.020Crossref PubMed Scopus (97) Google Scholar, 2Wang J.C. Gray N.E. Kuo T. Harris C. Regulation of triglyceride metabolism by glucocorticoid receptor.Cell Biosci. 2012; 2 (22640645): 1910.1186/2045-3701-2-19Crossref PubMed Scopus (81) Google Scholar3de Guia R.M. Herzig S. How do glucocorticoids regulate lipid metabolism?.Adv. Exp. Med. Biol. 2015; 872 (26215993): 127-14410.1007/978-1-4939-2895-8_6Crossref PubMed Scopus (29) Google Scholar). In contrast, reducing glucocorticoid levels, such as inhibiting 11β-hydroxysteroid dehydrogenase type 1, an enzyme converting inactive glucocorticoids to active hormones, reduces plasma and liver TG levels in animal models of metabolic syndrome (4Hermanowski-Vosatka A. Balkovec J.M. Cheng K. Chen H.Y. Hernandez M. Koo G.C. Le Grand C.B. Li Z. Metzger J.M. Mundt S.S. Noonan H. Nunes C.N. Olson S.H. Pikounis B. Ren N. et al.11β-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice.J. Exp. Med. 2005; 202 (16103409): 517-52710.1084/jem.20050119Crossref PubMed Scopus (339) Google Scholar5Anil T.M. Dandu A. Harsha K. Singh J. Shree N. Kumar V.S. Lakshmi M.N. Sunil V. Harish C. Balamurali G.V. Naveen Kumar B.S. Gopala A.S. Pratibha S. Sadasivuni M. Anup M.O. et al.A novel 11β-hydroxysteroid dehydrogenase type1 inhibitor CNX-010-49 improves hyperglycemia, lipid profile and reduces body weight in diet induced obese C57B6/J mice with a potential to provide cardio protective benefits.BMC Pharmacol. Toxicol. 2014; 15 (25098735): 4310.1186/2050-6511-15-43Crossref PubMed Scopus (14) Google Scholar, 6Li G. Hernandez-Ono A. Crooke R.M. Graham M.J. Ginsberg H.N. Effects of antisense-mediated inhibition of 11β-hydroxysteroid dehydrogenase type 1 on hepatic lipid metabolism.J. Lipid Res. 2011; 52 (21364201): 971-98110.1194/jlr.M013748Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar7Nuotio-Antar A.M. Hachey D.L. Hasty A.H. Carbenoxolone treatment attenuates symptoms of metabolic syndrome and atherogenesis in obese, hyperlipidemic mice.Am. J. Physiol. Endocrinol. Metab. 2007; 293 (17878220): E1517-E152810.1152/ajpendo.00522.2007Crossref PubMed Scopus (62) Google Scholar) and nonalcoholic fatty liver disease patients (8Stefan N. Ramsauer M. Jordan P. Nowotny B. Kantartzis K. Machann J. Hwang J.H. Nowotny P. Kahl S. Harreiter J. Hornemann S. Sanyal A.J. Stewart P.M. Pfeiffer A.F. Kautzky-Willer A. et al.Inhibition of 11β-HSD1 with RO5093151 for non-alcoholic fatty liver disease: a multicentre, randomised, double-blind, placebo-controlled trial.Lancet Diabetes Endocrinol. 2014; 2 (24795254): 406-41610.1016/S2213-8587(13)70170-0Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In fact, an inhibitor for 11β-hydroxysteroid dehydrogenase type 1 is currently under clinical trial phase 1b, which showed a modest but significant improvement of nonalcoholic fatty liver disease (NAFLD) 3The abbreviations used are: NAFLDnonalcoholic fatty liver diseaseshRNAsmall hairpin RNAACPD2-acetyl-1,3-cyclopentanedionePKCprotein kinase CGRglucocorticoid receptorDNLde novo lipogenesisTGtriglycerideDexdexamethasonePPARperoxisome proliferator-activated receptorqPCRquantitative PCRAAVadeno-associated virus. (8Stefan N. Ramsauer M. Jordan P. Nowotny B. Kantartzis K. Machann J. Hwang J.H. Nowotny P. Kahl S. Harreiter J. Hornemann S. Sanyal A.J. Stewart P.M. Pfeiffer A.F. Kautzky-Willer A. et al.Inhibition of 11β-HSD1 with RO5093151 for non-alcoholic fatty liver disease: a multicentre, randomised, double-blind, placebo-controlled trial.Lancet Diabetes Endocrinol. 2014; 2 (24795254): 406-41610.1016/S2213-8587(13)70170-0Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Antagonists for the glucocorticoid receptor (GR) also improve plasma lipid profiles (9Priyadarshini E. Anuradha C.V. Glucocorticoid antagonism reduces insulin resistance and associated lipid abnormalities in high-fructose-Fed mice.Can. J. Diabetes. 2017; 41 (27614803): 41-5110.1016/j.jcjd.2016.06.003Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 10Luz J.G. Carson M.W. Condon B. Clawson D. Pustilnik A. Kohlman D.T. Barr R.J. Bean J.S. Dill M.J. Sindelar D.K. Maletic M. Coghlan M.J. Indole glucocorticoid receptor antagonists active in a model of dyslipidemia act via a unique association with an agonist binding site.J. Med. Chem. 2015; 58 (26218343): 6607-661810.1021/acs.jmedchem.5b00736Crossref PubMed Scopus (24) Google Scholar). Despite these long-established observations, the mechanisms governing these glucocorticoid effects are not entirely clear. Chronic glucocorticoid exposure has been shown to induce de novo lipogenesis (DNL) and triglyceride (TG) synthesis in liver (11Dolinsky V.W. Douglas D.N. Lehner R. Vance D.E. Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone.Biochem. J. 2004; 378 (14662008): 967-97410.1042/bj20031320Crossref PubMed Scopus (106) Google Scholar, 12Berdanier C.D. Role of glucocorticoids in the regulation of lipogenesis.Faseb J. 1989; 3 (2666232): 2179-218310.1096/fasebj.3.10.2666232Crossref PubMed Scopus (86) Google Scholar). However, treating hepatocytes with glucocorticoids alone does not always promote lipogenesis (13Amatruda J.M. Danahy S.A. Chang C.L. The effects of glucocorticoids on insulin-stimulated lipogenesis in primary cultures of rat hepatocytes.Biochem. J. 1983; 212 (6347191): 135-14110.1042/bj2120135Crossref PubMed Scopus (64) Google Scholar, 14Nasiri M. Nikolaou N. Parajes S. Krone N.P. Valsamakis G. Mastorakos G. Hughes B. Taylor A. Bujalska I.J. Gathercole L.L. Tomlinson J.W. 5α-Reductase type 2 regulates glucocorticoid action and metabolic phenotype in human hepatocytes.Endocrinology. 2015; 156 (25974403): 2863-287110.1210/en.2015-1149Crossref PubMed Scopus (32) Google Scholar). Thus, the induction of hepatic DNL and TG synthesis by chronic glucocorticoid exposure may require additional signals or the effects of glucocorticoids on other tissues. nonalcoholic fatty liver disease small hairpin RNA 2-acetyl-1,3-cyclopentanedione protein kinase C glucocorticoid receptor de novo lipogenesis triglyceride dexamethasone peroxisome proliferator-activated receptor quantitative PCR adeno-associated virus. It has been proposed that the excess lipolysis induced by GC in white adipose tissue plays a role in the development of dyslipidemia and fatty liver (15Arnaldi G. Scandali V.M. Trementino L. Cardinaletti M. Appolloni G. Boscaro M. Pathophysiology of dyslipidemia in Cushing's syndrome.Neuroendocrinology. 2010; 92 (20829625): 86-9010.1159/000314213Crossref PubMed Scopus (131) Google Scholar, 16Seckl J.R. Morton N.M. Chapman K.E. Walker B.R. Glucocorticoids and 11β-hydroxysteroid dehydrogenase in adipose tissue.Recent Prog. Horm. Res. 2004; 59 (14749510): 359-39310.1210/rp.59.1.359Crossref PubMed Scopus (199) Google Scholar). This proposed mechanism is in agreement with previous observations showing that glucocorticoid-induced hepatic steatosis and hypertriglyceridemia are dependent on the presence of Angptl4 (angiopoietin-like 4), a GR primary target gene (17Koliwad S.K. Kuo T. Shipp L.E. Gray N.E. Backhed F. So A.Y. Farese Jr, R.V. Wang J.C. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-regulated triglyceride metabolism.J. Biol. Chem. 2009; 284 (19628874): 25593-2560110.1074/jbc.M109.025452Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Angptl4 encodes a secreted protein that can promote intracellular lipolysis in adipocytes (18Gray N.E. Lam L.N. Yang K. Zhou A.Y. Koliwad S. Wang J.C. Angiopoietin-like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes.J. Biol. Chem. 2012; 287 (22267746): 8444-845610.1074/jbc.M111.294124Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). It has been shown that glucocorticoid-stimulated lipolysis in white adipose tissue is attenuated in mice lacking Angptl4 (Angptl4−/−) and purified ANGPTL4 protein directly induces lipolysis in mouse primary adipocytes (18Gray N.E. Lam L.N. Yang K. Zhou A.Y. Koliwad S. Wang J.C. Angiopoietin-like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes.J. Biol. Chem. 2012; 287 (22267746): 8444-845610.1074/jbc.M111.294124Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Thus, it is conceivable that Angptl4 mediates glucocorticoid stimulation of lipolysis in white adipose tissue, which mobilizes fatty acids to the liver for TG synthesis and storage. Notably, Angptl4 has also been shown to be required for glucocorticoids to stimulate hepatic ceramide production, which activates protein phosphatase 2A and protein kinase Cζ (PKCζ) to suppress insulin signaling in liver (19Chen T.C. Benjamin D.I. Kuo T. Lee R.A. Li M.L. Mar D.J. Costello D.E. Nomura D.K. Wang J.C. The glucocorticoid-Angptl4-ceramide axis induces insulin resistance through PP2A and PKCζ.Sci. Signal. 2017; 10 (28743803)eaai7905 10.1126/scisignal.aai7905Crossref PubMed Scopus (24) Google Scholar). Because hepatic ceramide levels have been positively associated with NAFLD (20Pagadala M. Kasumov T. McCullough A.J. Zein N.N. Kirwan J.P. Role of ceramides in nonalcoholic fatty liver disease.Trends Endocrinol Metab. 2012; 23 (22609053): 365-37110.1016/j.tem.2012.04.005Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 21Kasumov T. Li L. Li M. Gulshan K. Kirwan J.P. Liu X. Previs S. Willard B. Smith J.D. McCullough A. Ceramide as a mediator of non-alcoholic fatty liver disease and associated atherosclerosis.PLoS One. 2015; 10 (25993337)e0126910 10.1371/journal.pone.0126910Crossref PubMed Scopus (134) Google Scholar22Kurek K. Piotrowska D.M. Wiesiolek-Kurek P. Lukaszuk B. Chabowski A. Górski J. Zendzian-Piotrowska M. Inhibition of ceramide de novo synthesis reduces liver lipid accumulation in rats with nonalcoholic fatty liver disease.Liver Int. 2014; 34 (24106929): 1074-108310.1111/liv.12331Crossref PubMed Scopus (100) Google Scholar), we hypothesize that glucocorticoid-induced Angptl4-dependent hepatic production or ceramides are involved in the development of hepatic steatosis and hypertriglyceridemia. In this study, we examined this model and also identified the downstream effectors of ceramide-mediated signaling that mediate glucocorticoid response on hepatic TG homeostasis. Male WT and Angptl4−/− mice were treated with or without dexamethasone (a synthetic glucocorticoid) for 7 days. At the end of the treatment, blood and liver were collected, and plasma and liver TG levels were measured. Dexamethasone treatment significantly elevated plasma and liver TG levels in WT mice (Fig. 1, A and B). These dexamethasone effects were suppressed in Angptl4−/− mice (Fig. 1, A and B). To examine whether dexamethasone treatment stimulates hepatic DNL and TG synthesis and the role of Angptl4 in these processes, we applied a stable isotope labeling technique. In the liver of PBS-treated WT mice, the absolute rate of DNL (mg palmitate synthesized over a 24-h labeling period) was 0.08 mg palmitate/day/mouse (Fig. 1C). Dexamethasone treatment for 7 days resulted in a 40-fold induction in the absolute DNL rate (3.2 mg palmitate/day; Fig. 1C). In contrast, the absolute rate of DNL in the liver of untreated Angptl4−/− mice was 0.09 mg palmitate/day (Fig. 1C). Dexamethasone treatment increased the rate of DNL 2.4-fold (to 0.22 mg palmitate/day; Fig. 1C). This induction was markedly lower than the dexamethasone response in liver of WT mice (p < 0.05). These results demonstrate that Angptl4 is involved chronic dexamethasone exposure–stimulated hepatic DNL. Dexamethasone treatment also resulted in 6-fold induction of the absolute rate of TG synthesis in the liver of WT mice (Fig. 1D). The absolute TG synthesis rate in liver of untreated Angptl4−/− mice was not statistically significantly different from untreated WT mice (Fig. 1D). Dex treatment did not elevate the absolute hepatic TG synthesis rate in Angptl4−/− mice (Fig. 1D). These results demonstrate that without Angptl4, the ability of Dex to augment TG synthesis in the liver was diminished. We analyzed the expression of genes involved in DNL and TG synthesis in the liver of WT and Angptl4−/− mice treated with or without dexamethasone. We found that the expression of genes encoding enzymes in DNL and TG synthesis, such as Fasn, Acaca, Acacb, Dgat2, Gpat1, and Lpin1, were increased by dexamethasone in WT mice liver (Fig. 2A). In Angptl4−/− mice, the ability of Dex to activate these genes was significantly reduced (Fig. 2A). Interestingly, dexamethasone treatment significantly reduced the expression of Srebp1c, a transcription factor that activates the transcription of many lipogenic genes (23Jeon T.I. Osborne T.F. SREBPs: metabolic integrators in physiology and metabolism.Trends Endocrinol Metab. 2012; 23 (22154484): 65-7210.1016/j.tem.2011.10.004Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 24Wang Y. Viscarra J. Kim S.J. Sul H.S. Transcriptional regulation of hepatic lipogenesis.Nat. Rev. Mol. Cell Biol. 2015; 16 (26490400): 678-68910.1038/nrm4074Crossref PubMed Scopus (384) Google Scholar), in WT mice liver (Fig. 2A). In Angptl4−/− mice, the reduction of Srebp1c expression by dexamethasone was even more profound (Fig. 2A). In contrast, the expression of Chrebpβ, another transcription factor that augments lipogenic gene transcription (25Herman M.A. Peroni O.D. Villoria J. Schön M.R. Abumrad N.A. Blüher M. Klein S. Kahn B.B. A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism.Nature. 2012; 484 (22466288): 333-33810.1038/nature10986Crossref PubMed Scopus (416) Google Scholar), was markedly enhanced in WT but not Angptl4−/− mice liver (Fig. 2A). The expression of Chrebpα, a longer isoform of Chrebp (24Wang Y. Viscarra J. Kim S.J. Sul H.S. Transcriptional regulation of hepatic lipogenesis.Nat. Rev. Mol. Cell Biol. 2015; 16 (26490400): 678-68910.1038/nrm4074Crossref PubMed Scopus (384) Google Scholar, 26Baraille F. Planchais J. Dentin R. Guilmeau S. Postic C. Integration of ChREBP-mediated glucose sensing into whole body metabolism.Physiology (Bethesda). 2015; 30 (26525342): 428-43710.1152/physiol.00016.2015Crossref PubMed Scopus (36) Google Scholar), however, was not affected by dexamethasone (Fig. 2A). However, the expression of Chrebpα was still reduced in Angptl4−/− mice liver (Fig. 2A). The fact that Srebp1c expression was reduced by chronic dexamethasone exposure raised a question regarding whether Srebp1c was activated to promote lipogenic and TG synthetic gene transcription. Interestingly, protein levels of the mature and the immature forms of Srebp1c in the whole cell extracts and the mature form of Srebp1c in the nuclear extracts isolated from control and dexamethasone-treated liver were similar (Fig. 2B). These results demonstrated that despite the reduction of Srebp1c mRNA by dexamethasone treatment, Srebp1c protein levels were not significantly decreased by dexamethasone treatment for the time period conducted in our experiments. We performed ChIP experiments to further examine the recruitment of Srebp1c to its binding site in the Fasn gene. We found that Srebp1c was recruited to the Fasn gene promoter without dexamethasone treatment, which likely provides the basal lipogenic action (Fig. 2C). Dexamethasone treatment further enhanced the Srebp1c recruitment to the Fasn gene promoter (Fig. 2C). These results are in agreement with the increased lipogenic rate in the livers of dexamethasone-treated mice. We previously showed that chronic dexamethasone exposure increased the accumulation of various species of ceramides in the liver of WT mice, and these dexamethasone effects were attenuated in the liver of Angptl4−/− mice (19Chen T.C. Benjamin D.I. Kuo T. Lee R.A. Li M.L. Mar D.J. Costello D.E. Nomura D.K. Wang J.C. The glucocorticoid-Angptl4-ceramide axis induces insulin resistance through PP2A and PKCζ.Sci. Signal. 2017; 10 (28743803)eaai7905 10.1126/scisignal.aai7905Crossref PubMed Scopus (24) Google Scholar). To examine whether ceramides are involved in dexamethasone-induced hepatic steatosis and hypertriglyceridemia, WT and Angptl4−/− mice were treated with dexamethasone, and with or without myriocin, an inhibitor of serine palmitoyltransferase 1 and 2 (Spt1/2), rate-controlling enzymes in the de novo ceramide synthesis pathway (27Miyake Y. Kozutsumi Y. Nakamura S. Fujita T. Kawasaki T. Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin.Biochem. Biophys. Res. Commun. 1995; 211 (7794249): 396-40310.1006/bbrc.1995.1827Crossref PubMed Scopus (457) Google Scholar). Dexamethasone treatment was for 7 days, whereas myriocin was included in the final 4 days. Myriocin treatment decreased TG levels in both the plasma and the liver of dexamethasone-treated WT mice (Fig. 3, A and B). The TG levels in liver of dexamethasone-treated Angptl4−/− mice were significantly lower than those of dexamethasone-treated WT mice (Fig. 3, A and B). However, in dexamethasone-treated Angptl4−/− mice, myriocin did not further reduce the TG levels in plasma and liver (Fig. 3, A and B). These results suggest that increased ceramide production plays a key role in Angptl4 function in dexamethasone-induced TG accumulation in the plasma and the liver. We next monitored the effect of myriocin on the expression of lipogenic and TG synthetic genes in the liver of myriocin-treated WT and Angptl4−/− mice. We found that the expression of lipogenic genes, such as Fasn, Acaca, and Chrebpα, was reduced in the liver of myriocin-treated WT mice (Fig. 3C). The expression of Srebp1c, Cd36, and Chrebpβ, although not statistically significant, was also trending lower in myriocin-treated WT mice (Fig. 3C). Myriocin treatment did not affect the expression of these genes in Angptl4−/− mice. These results suggest that ceramide production is required for Angptl4’s role in the induction of lipogenic gene expression by chronic dexamethasone exposure. Interestingly, the expression of TG synthetic genes, such as Dgat2, Gpat1, and Lpin1, was not affected by myriocin treatment. We also found that myriocin increased the expression of Angptl4 in the liver but not epididymal white adipose tissue of WT mice treated with dexamethasone (Fig. 3D). To further confirm that the hepatic ceramide synthetic pathway is involved in triglyceride accumulation in the plasma and liver, we infected WT mice with adeno-associated virus (serotype 8) expressing scramble (control) small hairpin RNA (shRNA) or shRNA targeting Sptlc2, which encodes a rate-controlling enzyme of de novo ceramide synthesis. These mice were called AAV8-shSCR and AAV8-shSptlc2, respectively. We found that hepatic Sptlc2 expression was significantly lower in the liver of AAV8-shSptlc2 mice (Fig. 3E). Moreover, upon dexamethasone treatment, plasma and liver triglyceride levels were also significantly lower in AAV8-shSptlc2 mice than those of AAV8-shSCR mice (Fig. 3, F and G. These results are similar to those of the myriocin treatment experiments above (Fig. 3, A and B). PKCζ and PP2A are two known downstream effectors of ceramides. To examine whether PP2A is involved in glucose intolerance induced by glucocorticoids, WT mice were infected with adenovirus expressing scrambled shRNA or shRNA targeting Ppp2ca, which encodes the PP2A catalytic subunit. These mice were called Ad-shSCR and Ad-shPpp2ca, respectively. Western blots validated a significant reduction in the protein abundance of Ppp2ca by shRNA in mouse liver (Fig. 4A). However, we did not observe significant differences in plasma and liver TG levels between dexamethasone-treated Ad-shSCR and Ad-shPpp2ca mice (Fig. 4, B and C). To test the role of PKCζ in glucocorticoid-induced hepatic hypertriglyceridemia, WT and Angptl4−/− mice were injected with or without 2-acetyl-1,3-cyclopentanedione (ACPD), an inhibitor of atypical PKC, PKCζ, and PKCι (28Sajan M.P. Ivey R.A. Lee M.C. Farese R.V. Hepatic insulin resistance in ob/ob mice involves increases in ceramide, aPKC activity, and selective impairment of Akt-dependent FoxO1 phosphorylation.J. Lipid Res. 2015; 56 (25395359): 70-8010.1194/jlr.M052977Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 29Xia J.Y. Holland W.L. Kusminski C.M. Sun K. Sharma A.X. Pearson M.J. Sifuentes A.J. McDonald J.G. Gordillo R. Scherer P.E. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.Cell Metab. 2015; 22 (26190650): 266-27810.1016/j.cmet.2015.06.007Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) for the final 4 days of the 7-day dexamethasone course. Western blots showed that ACPD treatment reduced the levels of phosphorylated PKCζ in liver (Fig. 5A), which indicates reduced PKCζ activity. We found that ACPD treatment reduced both plasma and liver TG accumulation in WT but not Angptl4−/− mice (Fig. 5, B and C). Based on these results, PKCζ but not PP2A is involved in chronic dexamethasone-induced hepatic steatosis and hypertriglyceridemia. To confirm whether PKCζ is downstream of ceramides, we performed Western blots to monitor phosphorylated PKCζ levels in the liver of dexamethasone-treated WT and Angptl4−/− mice that were treated with or without myriocin. We found that the relative ratio of phosphorylated PKCζ and total phosphorylated PKCζ was reduced in liver of WT mice (Fig. 5D). These results confirmed that ceramide production is involved in chronic dexamethasone treatment-induced PKCζ activity. We also found that Angptl4 expression was decreased by ACPD treatment in epididymal white adipose tissue but not liver of WT mice (Fig. 5E). PPARα has been previously implicated in the production of ceramides in various cell types (30Baranowski M.B.A. Zabielski P. Górski J. PPARα agonist induces the accumulation of ceramide in the heart of rats fed high-fat diet.J. Physiol. Pharmacol. 2007; 58 (17440226): 57-72PubMed Google Scholar, 31Aye I.L. Gao X. Weintraub S.T. Jansson T. Powell T.L. Adiponectin inhibits insulin function in primary trophoblasts by PPAR-mediated ceramide synthesis.Mol. Endocrinol. 2014; 28 (24606127): 512-52410.1210/me.2013-1401Crossref PubMed Scopus (56) Google Scholar32Rivier M. Castiel I. Safonova I. Ailhaud G. Michel S. Peroxisome proliferator-activated receptor-α enhances lipid metabolism in a skin equivalent model.J. Invest. Dermatol. 2000; 114 (10733673): 681-68710.1046/j.1523-1747.2000.00939.xAbstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Moreover, PPARα has been shown to be involved in glucocorticoid-induced hepatic gluconeogenesis and insulin resistance (33Bernal-Mizrachi C. Weng S. Feng C. Finck B.N. Knutsen R.H. Leone T.C. Coleman T. Mecham R.P. Kelly D.P. Semenkovich C.F. Dexamethasone induction of hypertension and diabetes is PPAR-α dependent in LDL receptor-null mice.Nat. Med. 2003; 9 (12847522): 1069-107510.1038/nm898Crossref PubMed Scopus (175) Google Scholar). We therefore examine whether PPARα is involved in dexamethasone treatment–induced plasma and liver triglyceride accumulation. WT mice treated with dexamethasone were treated with PBS or a PPARα antagonist GW-6471. GW-6471 treatment improved glucose tolerance in dexamethasone-treated mice (Fig. 6A). These results were in agreement with the previous observation that showed the improvement of dexamethasone-regulated glucose homeostasis in PPARα knockout mice (33Bernal-Mizrachi C. Weng S. Feng C. Finck B.N. Knutsen R.H. Leone T.C. Coleman T. Mecham R.P. Kelly D.P. Semenkovich C.F. Dexamethasone induction of hypertension and diabetes is PPAR-α dependent in LDL receptor-null mice.Nat. Med. 2003; 9 (12847522): 1069-107510.1038/nm898Crossref PubMed Scopus (175) Google Scholar). However, GW-6471 treatment did not affect the accumulation of plasma and liver triglyceride levels in mice under dexamethasone treatment (Fig. 6, B and C). Dyslipidemia is one of the major adverse effects caused by chronic and/or excess glucocorticoid exposure. However, the mechanisms underlying this alteration are mostly unclear. Here we showed that dexamethasone treatment for 7 days elevated hepatic DNL and TG synthesis. Interestingly, previous studies show that glucocorticoid treatment alone on primary hepatocytes or hepatoma cells usually does not promote lipogenesis and/or TG synthesis (11Dolinsky V.W. Douglas D.N. Lehner R. Vance D.E. Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone.Biochem. J. 2004; 378 (14662008): 967-97410.1042/bj20031320Crossref PubMed Scopus (106) Google Scholar, 12Berdanier C.D. Role of glucocorticoids in the regulation of lipogenesis.Faseb J. 1989; 3 (2666232): 2179-218310.1096/fasebj.3.10.2666232Crossref PubMed Scopus (86) Google Scholar). This suggests that glucocorticoid effects on cell types other than hepatocytes are required for the enhancement of hepatic DNL and TG synthesis. Here we showed that Angptl4-dependent hepatic ceramide production is involved in chronic dexamethasone treatment-induced hepatic steatosis and hypertriglyceridemia (Fig. 7). Angptl4 is a GR primary target gene encoding a secreted protein that inhibits lipoprotein lipase through its N-terminal coiled-coil domain (34Ge H. Cha J.Y. Gopal H. Harp C. Yu X. Repa J.J. Li C. Differential regulation and properties of angiopoietin-like proteins 3 and 4.J. Lipid Res. 2005; 46 (15863837): 1484-149010.1194/jlr.M500005-JLR200Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 35Yau M.H. Wang Y. Lam K.S. Zhang" @default.
• W2943383566 created "2019-05-09" @default.
• W2943383566 creator A5010475028 @default.
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• W2943383566 date "2019-06-01" @default.
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• W2943383566 title "An ANGPTL4–ceramide–protein kinase Cζ axis mediates chronic glucocorticoid exposure–induced hepatic steatosis and hypertriglyceridemia in mice" @default.
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• W2943383566 doi "https://doi.org/10.1074/jbc.ra118.006259" @default.
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